Name: recording gap junction current from xenopus oocytes
Uploaded: 2022-01-21T14:15:00
Description: Watch this Scientific Journal Video about Recording Gap Junction Current from Xenopus Oocytes at JoVE.com

We thank Haiying Zhan, Qian Ge for their involvement in the initial stage of technical development, Kiranmayi Vedantham for helping with the figures, and Dr. Camillo Peracchia for advice on the oocyte pairing chamber.

The surgeries are performed following a protocol approved by the institutional animal care committee of the University of Connecticut School of Medicine.
1. Frog surgery and preparation of defollicuated oocytes
<ol>
	<li>Anesthetize an adult female African clawed frog (Xenopus laevis) (Table of Materials) by immersing in a cool (with ice) tricaine solution (~300 mg/L).</li>
	<li>Wait (~15 min) until the frog shows little or no response to squeezing ...

<ol>
	<li>Oshima, A., Matsuzawa, T., Murata, K., Tani, K., Fujiyoshi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hexadecameric+structure+of+an+invertebrate+gap+junction+channel.">Hexadecameric structure of an invertebrate gap junction channel.</a> Journal of Molecular Biology. 428 (6), 1227-1236 (2016).</li><li>Maeda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+connexin+26+gap+junction+channel+at+3.5+A+resolution.">Structure of the connexin 26 gap junction channel at 3.5 A resolution.</a> Nature. 458 (7238), 597-602 (2009).</li><li>Flores, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin-46/50+in+a+dynamic+lipid+environment+resolved+by+CryoEM+at+1.9+A.">Connexin-46/50 in a dynamic lipid environment resolved by CryoEM at 1.9 A.</a> Nature Communications. 11 (1), 4331 (2020).</li><li>Sohl, G., Willecke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions+and+the+connexin+protein+family.">Gap junctions and the connexin protein family.</a> Cardiovascular Research. 62 (2), 228-232 (2004).</li><li>Starich, T., Sheehan, M., Jadrich, J., Shaw, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innexins+in+C.+elegans.">Innexins in C. elegans.</a> Cell Communication &amp; Adhesion. 8 (4-6), 311-314 (2001).</li><li>Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innexins:+members+of+an+evolutionarily+conserved+family+of+gap-junction+proteins.">Innexins: members of an evolutionarily conserved family of gap-junction proteins.</a> Biochimica et Biophysica Acta. 1711 (2), 225-245 (2005).</li><li>Shui, Y., Liu, P., Zhan, H., Chen, B., Wang, Z. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+junctional+current+rectification+at+an+electrical+synapse.">Molecular basis of junctional current rectification at an electrical synapse.</a> Science Advances. 6 (27), (2020).</li><li>Starich, T. A., Xu, J., Skerrett, I. M., Nicholson, B. J., Shaw, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+innexins+UNC-7+and+UNC-9+mediate+electrical+synapse+specificity+in+the+Caenorhabditis+elegans+locomotory+nervous+system.">Interactions between innexins UNC-7 and UNC-9 mediate electrical synapse specificity in the Caenorhabditis elegans locomotory nervous system.</a> Neural Development. 4, 16 (2009).</li><li>Chen, B., Liu, Q., Ge, Q., Xie, J., Wang, Z. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+dependence+of+junctional+conductance+in+early+amphibian+embryos.">Voltage dependence of junctional conductance in early amphibian embryos.</a> Science. 204 (4391), 432-434 (1979).</li><li>Spray, D. C., Harris, A. L., Bennett, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equilibrium+properties+of+a+voltage-dependent+junctional+conductance.">Equilibrium properties of a voltage-dependent junctional conductance.</a> The Journal of General Physiology. 77 (1), 77-93 (1981).</li><li>Qu, Y., Dahl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+voltage+gate+of+gap+junction+channels:+selective+exclusion+of+molecules.">Function of the voltage gate of gap junction channels: selective exclusion of molecules.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (2), 697-702 (2002).</li><li>Swenson, K. I., Jordan, J. R., Beyer, E. C., Paul, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+gap+junctions+by+expression+of+connexins+in+Xenopus+oocyte+pairs.">Formation of gap junctions by expression of connexins in Xenopus oocyte pairs.</a> Cell. 57 (1), 145-155 (1989).</li><li>Tong, J. J., Liu, X., Dong, L., Ebihara, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exchange+of+gating+properties+between+rat+cx46+and+chicken+cx45.6.">Exchange of gating properties between rat cx46 and chicken cx45.6.</a> Biophysical Journal. 87 (4), 2397-2406 (2004).</li><li>Landesman, Y., White, T. W., Starich, T. A., Shaw, J. E., Goodenough, D. A., Paul, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innexin-3+forms+connexin-like+intercellular+channels.">Innexin-3 forms connexin-like intercellular channels.</a> Journal of Cell Science. 112, 2391-2396 (1999).</li><li>Skerrett, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+the+Xenopus+oocyte+expression+system+to+the+analysis+of+gap+junction+proteins.">Applying the Xenopus oocyte expression system to the analysis of gap junction proteins.</a> Methods in Molecular Biology. 154, 225-249 (2001).</li><li>Nielsen, P. A., Beahm, D. L., Giepmans, B. N., Baruch, A., Hall, J. E., Kumar, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning,+functional+expression,+and+tissue+distribution+of+a+novel+human+gap+junction-forming+protein,+connexin-31.9.+Interaction+with+zona+occludens+protein-1.">Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1.</a> Journal of Biological Chemistry. 277 (41), 38272-38283 (2002).</li><li>Kotsias, B. A., Salim, M., Peracchia, L. L., Peracchia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+cystic+fibrosis+transmembrane+regulator+and+gap+junction+channels+made+of+connexins+45,+40,+32+and+50+expressed+in+oocytes.">Interplay between cystic fibrosis transmembrane regulator and gap junction channels made of connexins 45, 40, 32 and 50 expressed in oocytes.</a> The Journal of Membrane Biology. 214 (1), 1-8 (2006).</li><li>Peracchia, C., Peracchia, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inversion+of+both+gating+polarity+and+CO2+sensitivity+of+voltage+gating+with+D3N+mutation+of+Cx50.">Inversion of both gating polarity and CO2 sensitivity of voltage gating with D3N mutation of Cx50.</a> American Journal of Physiology. 288 (6), 1381-1389 (2005).</li><li>del Corsso, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+mammalian+cells+with+connexins+and+measurement+of+voltage+sensitivity+of+their+gap+junctions.">Transfection of mammalian cells with connexins and measurement of voltage sensitivity of their gap junctions.</a> Nature Protocols. 1 (4), 1799-1809 (2006).</li><li>Peracchia, C., Wang, X. G., Peracchia, L. 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System optimization appears to be necessary for dual oocyte voltage-clamp experiments. Without it, recordings can be highly unstable, and the amplifiers may have to inject an excessive amount of current to reach the target Vm, resulting in oocyte damage and recording failures. Several factors are critical to obtaining stable dual oocyte recordings with the high side current measuring method. First, the current and voltage electrodes must have appropriate resistance (~1 M&#8486;), and their holders must be clean....

GJs are intercellular channels that may allow the current flow and exchange of small cytosolic molecules between neighboring cells. They exist in many cell types and perform diverse physiological functions. GJs in vertebrates are formed by connexins, whereas those in invertebrates by innexins. Each GJ consists of two juxtaposed hemichannels with either 6 or 8 subunits per hemichannel, depending on whether they are connexins or innexins1,2,3. Humans have 21 connexin genes4, while the commonly used invertebrate models C. elegans and Drosophila melanogaster have 25 and 8 innexin genes, respectively5,6. Alternative splicing of gene transcripts may further increase the diversity of GJ proteins, at least for innexins7,8.
GJs may be divided into three categories based on molecular compositions: homotypic, heterotypic, and heteromeric. A homotypic GJ has all its subunits being identical. A heterotypic GJ has two homomeric hemichannels, but the two hemichannels are formed by two different GJ proteins. A heteromeric GJ contains at least one heteromeric hemichannel. The molecular diversities of GJs may confer distinct biophysical properties that are important to their physiological functions. GJ biophysical properties are also modulated by regulatory proteins9. To understand how GJs perform their physiological functions, it is important to know their molecular compositions, biophysical properties, and the roles of regulatory proteins in their functions.
Heterologous expression systems are often used to study biophysical properties of ion channels, including GJs, and the effects of regulatory proteins on them. Because heterologous expression systems allow the expression of specific proteins, they are generally more amenable to dissecting protein functions than native tissues where proteins with redundant functions can complicate the analysis, and recording of Ij can be unattainable. Unfortunately, most commonly used cell lines except the Neuro-2A cell are inappropriate for studying GJ biophysical properties due to complications by endogenous connexins. Even Neuro-2A cells are not always appropriate for this kind of analysis. For example, we could not detect any Ij in Neuro-2A cells transfected with the innexins UNC-7 and UNC-9 in either the absence or the presence of UNC-1 (unpublished), which is required for the function of UNC-9 GJs in C. elegans9,10. On the other hand, Xenopus oocytes are a useful alternative system for electrophysiological analyses of GJs. Although they express an endogenous GJ protein, connexin 38 (Cx38)11, potential complications can be easily avoided by injecting a specific antisense oligonucleotide12. However, analyses of GJs with Xenopus oocytes require a differential voltage clamp of two juxtaposed cells, which is technically challenging. The earliest successes of double voltage clamp of frog blastomeres were reported about 40 years ago13,14. Since then, many studies have used this technique to record Ij in paired Xenopus oocytes. However, essentially all the previous studies have been performed with either homemade amplifiers12,15,16 or commercial amplifiers designed for recordings on single oocytes (GeneClamp 500, AxoClamp 2A, or AxoClamp 2B, Axon Instruments, Union City, CA)8,17,18,19,20. Because even the commercial amplifiers do not provide instructions for double oocyte voltage clamp, it is often challenging for new or less sophisticated electrophysiological labs to implement this technique.
Only one commercial amplifier has been developed for double oocyte voltage clamp, the OC-725C from Warner Instruments (Table of Materials, Figure 1A). This amplifier may be used in either a standard mode (for single oocytes) or a high side current measuring mode (for single or dual oocytes) depending on whether two sockets in its voltage probe are connected (Figure 1B, C). However, until our recent study7, there had not been a single publication describing the use of this amplifier in its high side current measuring mode. Although the amplifier has been used by another lab for dual oocyte recordings, it was used in the standard rather than the high side mode21,22. This lack of reports using the amplifier in its high side current measuring mode might be due to technical difficulties. We were unable to obtain stable dual oocyte recordings using the high side mode by following instructions from the manufacturer. Over the years, we have tried three different approaches for dual oocyte recordings, including using two OC-725C amplifiers in the high side current measuring mode, two OC-725C amplifiers in the standard mode, and two amplifiers from another manufacturer. We eventually succeeded in obtaining stable recordings only with the first approach after extensive trial and error. This publication describes and demonstrates the procedures we use to express GJ proteins in Xenopus oocytes, record Ij using the high side current measuring mode, and analyze the electrophysiological data using popular commercial software. Additional information about the double voltage-clamp technique may be found in other publications19,23.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar Bridge Magnetic Holder</td>
			<td>ALA Scientific Instruments</td>
			<td>MPSALT-H</td>
			<td>More stable than the Narishige tube clamper due to its larger magnetic base but it requires modification to accmmodate a 2-mm female socket.</td>
		</tr>
		<tr>
			<td>Auto Nanoliter Injector</td>
			<td>Drummond Scientific Company, Broomall, PA, USA</td>
			<td>Nanoject II</td>
			<td>Automated nanoliter injector</td>
		</tr>
		<tr>
			<td>Collagenase, Type II</td>
			<td>Gibco-USA, Langley, OK, USA</td>
			<td>17101-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond Scriber</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>62108-ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Differential Voltage Probe</td>
			<td>Warner Instruments, Hamden, CT, USA</td>
			<td>7255DI</td>
			<td></td>
		</tr>
		<tr>
			<td>Analog-to-Digital Signal Converter</td>
			<td>Molecular Devices, San Jose,CA, USA</td>
			<td>Digidata 1440A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Tweezers</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>500341</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>Drummond Scientific Company, Broomall, PA, USA</td>
			<td>3-000-203-G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot Wire Cutter</td>
			<td>Amazon.com</td>
			<td>Proxxon 37080</td>
			<td>An alternative is Hercules 8500 DHWT, which has a foot control pedal.</td>
		</tr>
		<tr>
			<td>Hyaluronidase, Type I-S</td>
			<td>MilliporeSigma, Burlington, MA, USA</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Holder Base</td>
			<td>Kanetec USA Corp. , Bensenville, IL, USA</td>
			<td>MB-L-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Beveler</td>
			<td>Sutter Instrument, Novato, CA, USA</td>
			<td>BV-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Holder</td>
			<td>World Precision Instruments, , Sarasota, FL, USA</td>
			<td>MEH1S15</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument, , Novato, CA, USA</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINETM T3</td>
			<td>Invitrogen-FisherScientific</td>
			<td>AM1348</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc MicroWell MiniTray</td>
			<td>Nalge Nunc International, Rochester, NY, USA</td>
			<td>438733</td>
			<td>Microwell Minitray</td>
		</tr>
		<tr>
			<td>Nylon mesh</td>
			<td>Component Supply Company, Sparta, TN, USA</td>
			<td>U-CMN-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Oocyte Clamp Amplifier</td>
			<td>Warner Instruments, , Hamden, CT, USA</td>
			<td>OC-725C</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro</td>
			<td>OriginLab Corporation, Northampton, MA, USA</td>
			<td>2020b</td>
			<td></td>
		</tr>
		<tr>
			<td>pClamp</td>
			<td>Molecular Devices, , San Jose,CA, USA</td>
			<td>Version 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Reference Electrode</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>DRIREF-2SH</td>
			<td>Specifications: https://www.wpiinc.com/blog/post/compare-dri-ref-reference-electrodes</td>
		</tr>
		<tr>
			<td>RNaseOUT (ribonuclease inhibitor)</td>
			<td>Invitrogen-FisherScientific</td>
			<td>10777-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk Suture 5-0</td>
			<td>Covidien, North Haven, CT, USA</td>
			<td>VS890</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer NanoDrop Lite</td>
			<td>Thermo Scientific</td>
			<td>ND-LITE-PR</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin Wall Glass Capallaries</td>
			<td>World Precision Instruments,Sarasota, FL, USA</td>
			<td>TW150F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube Clamper</td>
			<td>Narishige International USA, Amityville, NY, USA</td>
			<td>CAT-1</td>
			<td>Ready to use but its position is prone to shift due to the small magnetic base.</td>
		</tr>
		<tr>
			<td>Xenopus laevis</td>
			<td>Xenopus Express, Brooksville, FL, USA</td>
			<td>IMP-XL-FM</td>
			<td></td>
		</tr>
	</tbody>
</table>

recording gap junction current from xenopus oocytes

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions (GJs). However, this approach is technically challenging because it requires a differential voltage clamp of two opposed oocytes sharing a common ground. Although a small number of labs have succeeded in performing this technique, essentially all of them have used either homemade amplifiers or commercial amplifiers that were designed for single-oocyte recordings. It is often challenging for other labs to implement this technique. Although a high side current measuring mode has been incorporated into a commercial amplifier for dual oocyte voltage-clamp recordings, there had been no report for its application until our recent study. We have made the high side current measuring approach more practical and convenient by introducing several technical modifications, including the construction of a magnetically based recording platform that allows precise placement of oocytes and various electrodes, use of the bath solution as a conductor in voltage differential electrodes, adoption of a commercial low-leakage KCl electrode as the reference electrode, fabrication of current and voltage electrodes from thin-wall glass capillaries, and positioning of all the electrodes using magnetically based devices. The method described here allows convenient and robust recordings of junctional current (Ij) between two opposed Xenopus oocytes.

UNC-7 and UNC-9 are innexins of C. elegans. While UNC-9 has only one isoform, UNC-7 has multiple isoforms that differ mainly in the length and amino acid sequence of their amino terminals7,8. These innexins may form homotypic as well as heterotypic (of UNC-7 and UNC-9) GJs when expressed in Xenopus oocytes7,8. Representative Ij traces and the resulting normalized ...

Watch this Scientific Journal Video about Recording Gap Junction Current from Xenopus Oocytes at JoVE.com

Recording Gap Junction Current from Xenopus Oocytes

Here we present a protocol to express gap junction proteins in Xenopus oocytes and record junctional current between two apposed oocytes using a commercial amplifier designed for dual oocyte voltage-clamp recordings in a high side current measuring mode.

Recording Gap Junction Current from Xenopus Oocytes

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions ...

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for analyzing biophysical properties of gap junctions. The main advantage of our technique is to use a high-side current measuring approach that is appropriate for double site voltage clamps. The procedure will be demonstrated by Yuan Shui, a post-doctoral fellow from my laboratory.As soon as 70 to 80%of the oocytes are defoliculated, decant the enzyme solution by gently tilting the tube. Wash the oocytes five times by filling the tube with ND96 solution and then decanting the solution. After the final wash, transfer the oocytes to a 60-millimeter Petri dish containing ND96 solution.Use a clean glass Pasteur pipette to pick and transfer large, healthy-looking oocytes to a 60-millimeter Petri dish containing ND96 solution supplemented with sodium pyruvate and penicillin streptomycin. Prepare an oocyte injection dish by gluing a small piece of nylon mesh to the bottom of a 35-millimeter Petri dish. Fill the oocyte injection dish approximately halfway with ND96 solution supplemented with pyruvate and penicillin streptomycin.Then, place 25 to 30 oocytes in rows inside the Petri dish. Back fill an injection micropipette with lightweight mineral oil and insert it into the micropipette holder of the injector. Transfer the cRNA from one of the stored aliquots to the inside surface of a Petri dish cover or bottom by pipetting.Press the fill button in the injector controller to aspirate the cRNA droplet into the tip of the micropipette. Then, press the inject button to inject the cRNA. Transfer the injected oocytes to a new Petri dish containing ND96 solution supplemented with pyruvate and penicillin streptomycin.Keep them inside the environmental chamber at 15 to 18 degrees Celsius for one to three days. Replace the solution daily by transferring the oocytes to a new Petri dish. Use two fine tweezers to gently peel off the transparent villin membrane that wraps the oocyte.Then, place two oocytes per well in an oocyte pairing chamber containing the ND96 solution. Connect the differential voltage probe and the current cables to the model cell. Then, connect the red and black sockets to the differential voltage probe with the included jumper and connect either leg of the jumper to either pin in the model cell.Connect the ground wire in the model cell to the cable from the grounds circuit socket of the amplifier. Turn the VM offset and VE offset knobs to zero the membrane voltage and membrane current meters. Switch the clamp from off to either fast or slow and turn the gain dial clockwise to a level that allows proper voltage clamp to zero the voltage electrode and bath electrode meters of the amplifier.Run a simple acquisition protocol containing a few voltage steps to confirm that the voltage displayed in the membrane voltage meter changes according to the acquisition protocol, and that voltage and current traces are displayed properly in Clampex. Place a Petri dish containing the paired oocytes into the Petri dish receptacle of the recording platform. Select a pair of oocytes and rotate the Petri dish if necessary, so that the two oocytes are in the left and right direction.Lock the stage in position by its magnetic base. Place a reference electrode near the edge of the Petri dish towards the user side. Then, connect the reference electrode to the black socket in only one of the two differential voltage probes.Take a pair of glass micropipettes, break off a bit of the tip with a diamond scriber, and smooth the tip edge by fire polishing. Hold the micropipette above the flame of an alcohol burner and bend it to a smooth angle at approximately one centimeter away from the tip. Back fill the glass pipette completely with ND96 solution and then insert it into a pre-filled microelectrode holder.Ensure that no air bubbles are present in the system. Insert the two-millimeter pin of the microelectrode holder into the two-millimeter socket of a differential voltage electrode connection wire. Clamp the two-millimeter socket on a magnetically-based clamper and aim the tip of the differential voltage electrode toward one of the two oocytes.Insert the one-millimeter pin of the differential voltage electrode connection wire into the red socket of the differential voltage probe on the same side. Prepare the voltage and current electrodes from the glass capillaries and back fill them with potassium chloride solution. Then, insert the electrodes into the electrode holders provided with the amplifiers.Lower the electrodes into the bath solution and turn the VM offset and VE offset dials to zero the membrane voltage and membrane current meters. Check the electrode resistance by pressing VM electrode test and VE electrode test. Insert the current and voltage electrodes into the oocytes and observe the negative membrane potentials.Next, in the clamp section of the amplifier, confirm that DC gain is at the in position. Turn the gain knob clockwise to a level that allows proper voltage clamp and turn the clamp switch from off to fast. Finally, run an acquisition protocol.A diagram of the oocyte experiment is shown here. Negative and positive membrane voltage steps are applied to oocyte 1 from a holding membrane voltage of minus 30 millivolts, whereas oocyte 2 is held at a constant membrane voltage of minus 30 millivolts. The trans-junctional voltage, or Vj, is defined as the membrane voltage of oocyte 2 minus the membrane voltage of oocyte 1.The representative images show the sample Lj traces and the resulting normalized junctional conductance trans-junctional voltage relationship of UNC-9 homotypic gap junctions. Sample Lj traces and the resulting normalized Gj-Vj relationship of UNC-7b homotypic gap junctions are shown here. The results show that these two types of Gjs differ in the Vj-dependent Ij inactivation rate, Vj dependence, and the amount of residual Gj.While attempting the procedure, it's very important to make sure that the differential voltage electrode system does not have any air bubbles and its tip is aimed very close to the oocyte and the voltage and current electrodes have a resistance of approximately one microohm.Our technique is easy to implement. It may be of particular interest to those labs that are newly interested in using Xenopus oocytes to study gap junctions.

recording-gap-junction-current-from-xenopus-oocytes

Research

JoVE Journal

Biology

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...